ATP and DNA substrate structure influence the annealing activities of WRN-E84A and BLM. A) Labeled C80 (0.025 nM) was incubated for 15 min at 37°C with WRN-E84A (0.5–2.5 nM) and unlabeled G80 (0.025 nM) in the presence or absence of ATP (1 mM) as indicated. These reactions were analyzed as specified in Methods for the strand pairing assay that measures conversion of single-stranded reactants to duplex products (indicated here and subsequently by an upward arrow between DNA structures at left; asterisks show the positions of radiolabels). B) Similar experiments were performed as in A, except using BLM (1.2–6 nM). C) Reactions were performed and analyzed as in A, without or with WRN-E84A (0.8 nM) and varying amounts of ATP (0–4 mM) as indicated. D) Experiments were performed as in C, except with BLM (2.4 nM). For panels A-D, the percentage of protein-dependent annealing calculated for each enzyme-containing reaction is given above the relevant lanes. E) As in A, annealing experiments were performed for the indicated times with WRN-E84A (9 nM), except using unlabeled G95 (0.025 nM) as the complement for labeled C80. Quantitation and graphical representation of the WRN- and BLM-dependent formation of duplex product (including subtraction of protein-independent annealing) in the absence (■) and presence (●) of ATP is depicted at right. F) Similar experiments with BLM (3 nM) were performed and analyzed as described in E.